Multi-pathogen wastewater surveillance in Malawi: An assessment of culture-based, RT-PCR, and long-read metagenomics methods with multiple ongoing outbreaks.

There is little infrastructure for human disease testing apart from rapid malaria diagnostic tests in many low- and middle-income countries of sub-Saharan Africa outside major referral hospitals, and animal testing is also limited. Eight pathogens were studied in wastewater and environmental surveillance (WES) sites in Malawi with identified locally relevant disease prevalence: influenza A virus, influenza B virus, measles virus, respiratory syncytial virus, Salmonella Typhi, SARS-CoV-2, Vibrio cholerae, and yellow fever virus. Culture-based inoculation onto plates, single-plex RT-PCR, and/or Oxford Nanopore Technologies MinION genomics methods were used for a real-world assessment. This analysis included 20 grab samples from three locations over 4-weeks. Three samples tested positive for V. cholerae using culture-based methods, the only pathogen analyzed using this method. In 14 of the 20 samples, at least one pathogen was detected using PCR. Genomic analysis was conducted on 12 samples; V. cholerae was detected in eight samples, but Salmonella Typhi was not detected. V. cholerae was a clear case of disconnection between methods for the same sample during an outbreak period: 3/20 detected by culture, 1/20 detected by PCR (ctxAB), 3/20 detected by PCR (hlyA), and 8/12 detected by genomics. S. Typhi was the most frequently detected pathogen via PCR (10/20 detections) but was not detected by genomics. Influenza B, measles, SARS-CoV-2, and yellow fever viruses were determined to not be detected (Ct > 40) in the samples studied. This study demonstrated discrepancies in multi-pathogen WES detection across the three methods and highlights the need for further investigation into the limitations of their portability in local, low-resource, laboratories. This presents a valuable learning opportunity in regions experiencing ongoing outbreaks.